Method For Producing Carotenoids Each Having 50 Carbon Atoms

ABSTRACT

The present invention provides a method of producing a carotenoid having 50 carbon atoms which comprises culturing, in a medium, a cell transformed with a mutant phytoene desaturase gene and obtaining the carotenoid having 50 carbon atoms from the culture. The mutant phytoene desaturase gene has an introduced mutation to encode a mutant phytoene desaturase having an enhanced activity to desaturate a carotenoid backbone compound of 50 carbon atoms.

This application is a continuation of U.S. patent application Ser. No. 14/124,256, filed Dec. 6, 2013, which is a National Stage Application of PCT/JP2012/064817, filed Jun. 8, 2012, which claims priority from Japanese Patent Application No. 2011-130326, filed Jun. 10, 2011.

TECHNICAL FIELD

The present invention relates to a production method for a carotenoid having 50 carbon atoms, including the step of culturing, in a medium, a cell transformed with a mutant phytoene desaturase gene.

BACKGROUND ART

There exist various carotenoids in nature. Approximately 750 kinds of carotenoids have been identified heretofore, and many of the carotenoids have been shown to have useful physiological functions and industrial applicability such as an antioxidant activity or an antitumor activity, or a use as a functional pigment molecule. In general, carotenoids are compounds classified as tetraterpenes formed of an isoprene backbone of 30 or 40 carbon atoms. In nature, a linear backbone of 30 or 40 carbon atoms is formed as a basic backbone and then subjected to various modifications such as cyclization. Structural diversity of the carotenoids is attributed to diversity of such modifications. Further, carotenoids are known to have physiological activities greatly varying depending on structural differences based on the diversity of the modifications.

A large number of carotenoids have been obtained by isolation and extraction from nature such as plants, or by chemical synthesis. Recently, however, production utilizing microbial fermentation has also been performed. In order to establish biosynthetic pathways for rare carotenoids, which exist in nature in only trace amounts, and unnatural carotenoids, which do not exist in nature, research has been made by many researchers (Non Patent Literatures 1 to 8 and Patent Literature 1). In Non Patent Literatures 1 to 6, carotenoids having various structures have been obtained by synthesis based on the so-called combinatorial biosynthesis technique. The combinatorial biosynthesis is a technology involving altering biosynthetic pathways of microorganisms using a genetic engineering technique and allowing the microorganisms to produce a compound of interest. Modifying enzymes that provide various structures to carotenoids have the “locally specific” nature of recognizing and acting on only part of substrates. Based on such nature of the modified substrates, the combinatorial biosynthesis of the carotenoids has been performed (Non Patent Literature 9). Meanwhile, in Non Patent Literatures 7, 8, and 11 and Patent Literature 1, biosynthesis of various unnatural carotenoids has been achieved by a method involving constructing a metabolic pathway using an activity of an enzyme that cannot be found in nature and is created using protein engineering.

As described above, the carotenoids that exist in nature have backbones of 30 carbon atoms and 40 carbon atoms. The former is derived from 4,4′-diapophytoene, which is synthesized via head-to-head condensation of two molecules of farnesyl diphosphate (C₁₅PP). The latter is derived from phytoene (carotenoid backbone compound of 40 carbon atoms), which is synthesized via head-to-head condensation of two molecules of geranylgeranyl diphosphate (C₂₀PP). The former is a key component of biosynthetic pathways for 10-odd kinds of carotenoids known to exist in nature, and the latter is a key component of biosynthetic pathways for about 700 or more kinds of carotenoids.

Further, there is a report that a carotenoid having a backbone of 50 carbon atoms, which is larger than that of 40 carbon atoms, exists in nature (Patent Literature 2). The carotenoid having a backbone of 50 carbon atoms is synthesized by binding an isoprene unit to a carotenoid having 40 carbon atoms “as addition” so as to increase the total number of carbon atoms to 45 and 50 (Non Patent Literature 10). In a synthetic pathway for a backbone of 40 or more carbon atoms, such as a backbone of 50 carbon atoms or 60 carbon atoms, synthesis is performed by using, for example, geranylfarnesyl diphosphate (C₂₅PP) or hexaprenyl diphosphate (C₃₀PP) as a raw material. Although the carotenoid having a backbone of 40 or more carbon atoms is expected to have many potentialities for physiological and pigment functions and the like different from conventional ones, there is no detailed report on its synthetic pathways in nature, and there are few reports on its artificial biosynthetic pathways.

Dr. Umeno, one of the inventors of the present invention, developed an enzyme having a function of synthesizing a carotenoid backbone compound of 50 carbon atoms via condensation of two molecules of geranylfarnesyl diphosphate (C₂₅PP) by altering a synthase (CrtM) for a carotenoid having 30 carbon atoms derived from Staphylococcus aureus. In addition, Dr. Umeno succeeded for the first time in the world in co-expressing the enzyme with an appropriate precursor synthase in Escherichia coli to produce 16,16′-diisopentenylphytoene, a carotenoid backbone compound of 50 carbon atoms (Non Patent Literature 7). However, in the synthetic pathway in Non Patent Literature 7, carotenoids having backbones of, for example, 30 carbon atoms, 40 carbon atoms, and 45 carbon atoms were synthesized simultaneously with that having a backbone of 50 carbon atoms. Even when a wild-type phytoene desaturase was added in this pathway, about 75% of the carotenoid backbone compound of 50 carbon atoms still remained without being desaturated, resulting in poor synthetic efficiency (Non Patent Literature 8).

CITATION LIST Patent Literature

-   [PTL 1] US 2002/0051998 A -   [PTL 2] JP 07-132096 A

Non Patent Literature

-   [NPL 1] Takaichi S. et al., Eur J Biochem 241, 291-6 (1996) -   [NPL 2] Yokoyama A. et al., Tetrahedron Lett. 39, 3709-12 (1998) -   [NPL 3] Albrecht M. et al., Nat. Biotechnol. 18, 843-6 (2000) -   [NPL 4] Lee P. C. et al., Chem. Biol. 10, 453-462 (2003) -   [NPL 5] Mijts B. N. et al., Chem. Biol. 12, 453-460 (2005) -   [NPL 6] Umeno D. et al., Appl Environ Microbiol 69, 3573-3579 (2003) -   [NPL 7] Umeno D. et al., J Bacteriol, 186, 1531-1536 (2004) -   [NPL 8] Tobias A. V. et al., Biochim Biophys Acta, 1761, 235-246     (2006) -   [NPL 9] Umeno D. et al., Microbiol Mol Biol Rev 69, 51-78 (2005) -   [NPL 10] Krubasik P. et al., Eur J Biochem 268, 3702-3708 (2001) -   [NPL 11] Schmidt-Dannert C. et al., Nature Biotech. 18, 750-753     (2000)

SUMMARY OF INVENTION Technical Problem

A carotenoid having 50 carbon atoms has a large backbone as compared to a carotenoid having 40 carbon atoms, and hence has such superiority as a substance that conjugated double bonds having a large size can be accommodated. It is considered that, when a carotenoid backbone compound of 50 carbon atoms can be synthesized and desaturated as in the synthetic pathway for the carotenoid having 40 carbon atoms in nature, the resultant product can be used in combination with various modifying enzymes to produce carotenoids having 50 carbon atoms having a great variety of structures and physiological activities. An object of the present invention is to provide a production method for various carotenoids having 50 carbon atoms, including efficiently desaturating a carotenoid backbone compound of 50 carbon atoms.

Solution to Problem

The inventors of the present invention have made intensive studies in order to achieve the object. Consequently, the inventors have found that a carotenoid backbone compound of 50 carbon atoms can be efficiently desaturated with a mutant phytoene desaturase obtained by introducing a mutation into a phytoene desaturase (CrtI), and have focused attention on the fact that a carotenoid having 50 carbon atoms can be efficiently and simply synthesized by culturing a cell having introduced therein a mutant phytoene desaturase gene, thus achieving the present invention.

That is, the present invention relates to the following items.

1. A method of producing a carotenoid having 50 carbon atoms, comprising: culturing, in a medium, a cell transformed with a mutant phytoene desaturase gene; and

obtaining the carotenoid having 50 carbon atoms from a culture after the culturing,

wherein the mutant phytoene desaturase gene has an introduced mutation to encode a mutant phytoene desaturase having an enhanced activity to desaturate a carotenoid backbone compound of 50 carbon atoms. 2. The method of producing a carotenoid having 50 carbon atoms according to the above-described item 1, wherein

the mutant phytoene desaturase gene has an introduced mutation to encode a mutant phytoene desaturase having an enhanced activity to desaturate a carotenoid backbone compound of 50 carbon atoms has been introduced, and

the mutation causes a substitution of an amino acid corresponding to at least one amino acid selected from asparagine at position 304, phenylalanine at position 339, isoleucine at position 338, aspartic acid at position 395, and isoleucine at position 228 in an amino acid sequence set forth in SEQ ID NO: 1.

3. The method of producing a carotenoid having 50 carbon atoms according to the above-described item 1 or 2, wherein

the mutant phytoene desaturase gene has an introduced mutation to encode a mutant phytoene desaturase having an enhanced activity to desaturate a carotenoid backbone compound of 50 carbon atoms has been introduced, and

the mutation causes at least a substitution of an amino acid corresponding to asparagine at position 304 in SEQ ID NO: 1 by proline or serine.

4. The method of producing a carotenoid having 50 carbon atoms according to any one of the above-described items 1 to 3, wherein

the mutant phytoene desaturase gene has an introduced mutation to encode a mutant phytoene desaturase having an enhanced activity to desaturate a carotenoid backbone compound of 50 carbon atoms has been introduced, and

the mutant phytoene desaturase gene is obtained by introducing the mutation into a phytoene desaturase gene derived from Pantoea ananatis.

5. The method of producing a carotenoid having 50 carbon atoms according to any one of the above-described items 1 to 4, wherein the cell is Escherichia coli or yeast. 6. The method of producing a carotenoid having 50 carbon atoms according to any one of the above-described items 1 to 5, wherein the cell transformed with the mutant phytoene desaturase gene is further transformed with a gene encoding an enzyme that synthesizes the carotenoid backbone compound of 50 carbon atoms via condensation of two molecules of geranylfarnesyl diphosphate. 7. The method of producing a carotenoid having 50 carbon atoms according to any one of the above-described items 1 to 6, wherein the cell as defined in any one of the above-described items 1 to 6 is further transformed with a gene encoding an enzyme that synthesizes geranylfarnesyl diphosphate from farnesyl diphosphate and/or geranylgeranyl diphosphate. 8. The method of producing a carotenoid having 50 carbon atoms according to any one of the above-described items 1 to 7, wherein the cell as defined in any one of the above-described items 1 to 7 is further transformed with a gene encoding an enzyme that cyclizes ends of a desaturated carotenoid having 50 carbon atoms obtained by desaturating the carotenoid backbone compound of 50 carbon atoms. 9. The method of producing a carotenoid having 50 carbon atoms according to the above-described item 8, wherein

the cyclization as defined in the above-described item 8 comprises β-cyclization, and

the cell as defined in the above-described item 8 is further transformed with a gene encoding an enzyme that hydroxylates a β-ring and/or an enzyme that ketolates a β-ring in a carotenoid having 50 carbon atoms and having the β-ring at an end thereof.

10. The method of producing a carotenoid having 50 carbon atoms according to any one of the above-described items 1 to 7, wherein the cell as defined in any one of the above-described items 1 to 7 is further transformed with a gene encoding an enzyme that oxidizes a desaturated carotenoid having 50 carbon atoms obtained by desaturating the carotenoid backbone compound of 50 carbon atoms. 11. A mutant phytoene desaturase gene, into which a mutation to encode a mutant phytoene desaturase having an enhanced activity to desaturate a carotenoid backbone compound of 50 carbon atoms has been introduced. 12. The mutant phytoene desaturase gene according to the above-described item 11, wherein the mutation to encode a mutant phytoene desaturase having an enhanced activity to desaturate a carotenoid backbone compound of 50 carbon atoms causes a substitution of an amino acid corresponding to at least one amino acid selected from asparagine at position 304, phenylalanine at position 339, isoleucine at position 338, aspartic acid at position 395, and isoleucine at position 228 in an amino acid sequence set forth in SEQ ID NO: 1. 13. The mutant phytoene desaturase gene according to the above-described item 11 or 12, wherein the mutation of the mutant phytoene desaturase gene causes at least a substitution of an amino acid corresponding to asparagine at position 304 in SEQ ID NO: 1 by proline or serine. 14. A mutant phytoene desaturase, which is encoded by the mutant phytoene desaturase gene according to any one of the above-described items 11 to 13. 15. A cell producing a carotenoid having 50 carbon atoms by desaturating a carotenoid backbone compound of 50 carbon atoms, which is transformed with the mutant phytoene desaturase gene according to any one of the above-described items 11 to 13.

Advantageous Effects of Invention

The production method of the present invention allows the carotenoid backbone compound of 50 carbon atoms to be efficiently desaturated, and thus the carotenoids having 50 carbon atoms having a great variety of structures can be produced. Some enzymes including a wild-type phytoene desaturase exhibited a desaturation efficiency of only about several % at maximum for the carotenoid backbone compound of 50 carbon atoms. In contrast, the production method of the present invention has provided drastically increased desaturation efficiency. Thus, a desaturated carotenoid having 50 carbon atoms can be delivered to downstream modifying enzymes, and thereby a great variety of carotenoids having 50 carbon atoms can be produced.

A carotenoid backbone compound of 40 carbon atoms (30 carbon atoms in certain species of bacteria) free of oxygen and the like undergoes various modifications such as oxygenation. As a result, carotenoids acquire a great variety of structures. The most major carotene in nature is β-carotene having a β-ring, and various enzymes that modify the β-ring exist in nature. According to the production method of the present invention, a carotenoid having 50 carbon atoms and a β-ring can be synthesized. In addition, a great variety of carotenoids can be produced by using various enzymes that modify the β-ring.

The carotenoid having 50 carbon atoms produced by the production method of the present invention is expected to, for example, have the following potentialities: an antioxidant function improved as compared to a conventional carotenoid; a color gamut as a pigment extended to a range that has not been reported heretofore; and property of hardly undergoing destructive metabolism.

Further, in the production method of the present invention, conventional culture media and conditions may be used, and hence the carotenoid having 50 carbon atoms can be simply synthesized.

In addition, the production method of the present invention can be utilized for carrying out the highly efficient synthesis of a desaturated C₅₅ carotenoid, a desaturated C₆₀ carotenoid, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates functions of enzymes involved in carotenoid biosynthesis, and carotenoid biosynthetic pathways. Abbreviations for the enzymes that catalyze the corresponding reactions are given beside respective arrows. Further, numerals given in parentheses following compound names correspond to numbers of peaks of HPLC analysis in FIGS. 6A-6I.

FIGS. 2A-2H illustrate plasmid maps used in Examples 1 to 6 and Reference Examples 1 and 2. FIG. 2A illustrates pAC-fds_(Y81A,V157A)-crtM_(F26A,W38A,F233S), which was produced by inserting lac promoter/operator (lacPP)-crtM_(F26A,W38A,F233S) and lac promoter/operator (lacPO)-fds_(Y81A, V157A) into a pACmod vector. FIG. 2B illustrates pUC-pBAD-crtI*, which was produced by removing the lac promoter/operator from a pUC18Nm vector, inserting an araC gene/araBAD promoter sequence (derived from a pBADHisA vector), and inserting crtI variants (crtI*) downstream of the promoter. FIG. 2C illustrates pUC-pBAD-crtI*-crtY, which was produced by inserting crtY downstream of the crtI variants in pUC-pBAD-crtI*. FIG. 2D illustrates pUC-pBAD-crtI*-crtWZY, which was produced by inserting crtW, crtZ, and crtY downstream of the crtI variants in pUC-pBAD-crtI*. FIG. 2E illustrates pUC-pBAD-crtI*-crtA, which was produced by inserting crtA downstream of the crtI variants in pUC-pBAD-crtI*. FIG. 2F illustrates pUC-fds_(Y81A,V157A), which was produced by inserting an fds_(Y81A,V157A) gene downstream of lacPO in a pUC18Nm vector. FIG. 2G illustrates pAC-crtM_(F26A,W38A,F233S), which was produced by inserting lacPO-crtM_(F26A,W38A,F233S) into the BamHI site of a pACmod vector. FIG. 2H illustrates pAC-crtM_(F26A,W38A,F233S)-idi, which was produced by inserting lacPO-idi upstream of the ClaI site of pAC-crtM_(F26A,W38A,F233S).

FIG. 3 shows the synthesis amount of a C₅₀ carotenoid backbone compound in the case where Idi is co-expressed in a metabolic pathway constructed with FDS_(Y81A, V157A) and CrtM_(F26A, W38A, F233S) Example 1). “Absence of idi” and “Presence of idi” represent the synthesis amounts of a compound having a C₃₅ carotenoid backbone (35), a compound having a C₄₀ carotenoid backbone (40), a compound having a C₄₅ carotenoid backbone (45), and a compound having a C₅₀ carotenoid backbone (50) obtained by subjecting acetone extracts from E. coli transformed with various genes to HPLC analysis. Specifically, in FIG. 3, “35” represents 4-apophytoene, “40” represents phytoene, “45” represents 16-isopentenylphytoene, and “50” represents C₅₀-carotene (n=3).

FIGS. 4A and 4B illustrate the screening of a mutant phytoene desaturase capable of efficiently desaturating a carotenoid backbone compound of 50 carbon atoms (Example 2). FIG. 4A illustrates an overview of procedures of a screening experiment system, and FIG. 4B illustrates a screening principle.

FIGS. 5A and 5B show the actual colors of acetone extracts of 6 colonies out of 8 colonies obtained by screening, and the absorption spectra of the acetone extracts (Example 3). CrtEBI, a control, shows the result of the colony of E. coli transformed with a plasmid pAC-EBI containing crtE, crtB, and crtI derived from Pantoea ananatis. WT (or CrtIwt) shows the result of the colony of E. coli transformed with wild-type crtI, while mut1, mut2, mut8, mut4, and mut6 shows the results of the colonies of CrtI-m1, CrtI-m2, CrtI-m8, CrtI-m4, and CrtI-m6, respectively. It should be noted that the transformed E. coli synthesized lycopene.

FIGS. 6A-6I show the results of HPLC analysis of carotenoids produced by culturing E. coli that was co-transformed with pAC-fds_(Y81A,V157A)-crtM_(F26A,W38A,F233S) and various plasmids (Examples 3 to 6). FIGS. 6A to 6H show chromatograms obtained from HPLC analysis when introducing various plasmids. FIG. 6I shows the absorption spectra of compounds forming respective peaks. In FIG. 6I, the rightmost numerical values correspond to the numbers of the peaks in the chromatograms, and the three-digit numerical values are values for maximum absorption wavelengths.

FIG. 7 shows the results of β-cyclic carotenoid synthesis through the cooperation of CrtI with CrtY. The photographs show colony colors in the 0 to 48 hours culture of cells into which various genes have been introduced (Example 4). CrtI shows the case where wild-type crtI was introduced into E. coli, CrtIY shows the case where both of wild-type crtI and crtY were introduced into E. coli, CrtI₂ shows the case where a crtI variant derived from CrtI-m2 was introduced into E. coli, and CrtI₂Y shows the case where both of the crtI variant derived from CrtI-m2 and crtY were introduced into E. coli. It should be noted that in each E. coli pAC-fds_(Y81A,V157A)-crtM_(F26A,W38A,F233S) has been introduced.

FIGS. 8A-8C show the results of carotenoid synthesis through the cooperation of CrtI with CrtW, CrtZ, and CrtY, in FIG. 8A, and the results of carotenoid synthesis through the cooperation of CrtI with CrtA, in FIG. 8B (Example 5). FIGS. 8A and 8B show the absorption spectra of acetone extracts from E. coli transformed with various genes, and FIG. 8C is photographs showing the actual colors of cell pellets (upper row) and acetone extracts (lower row) in a similar case. CrtI* means that a crtI variant derived from CrtI-m2 was introduced into E. coli.

FIGS. 9A-9C show the absorption spectra of C₅₀-carotene (n=3), C₅₀-lycopene (n=15), and C₅₀-β-carotene, respectively in FIGS. 9A, 9B and 9C (Examples 3 and 4).

FIG. 10 shows the results of HPLC analysis of synthesis amounts of C₅₀-carotene (n=3), C₅₀-lycopene, and C₅₀-β-carotene by CrtI-m2 and CrtI_(N304P) (Example 7). (a), (b), (c), (d), and (e) show the results of E. coli transformed with pUC-pBAD-CrtI, E. coli transformed with pUC-pBAD-CrtI-m2, E. coli transformed with pUC-pBAD-CrtI_(N304P) , E. coli transformed with pUC-pBAD-CrtI-m2-CrtY, and E. coli transformed with pUC-pBAD-CrtI_(N304P)-CrtY, respectively. Only C₅₀-carotene (n=3) was synthesized in (a), C₅₀-carotene (n=3) and C₅₀-lycopene were synthesized in (b) and (c), and C₅₀-carotene (n=3) and C₅₀-β-carotene were synthesized in (d) and (e).

FIGS. 11A-11D illustrate plasmid maps used in Examples 8 and 9. The plasmids contain CrtW and/or CrtZ derived from Brevundimonas sp. strain SD-212. FIG. 11A illustrates pUC-pBAD-CrtI_(N304P)-CrtY-CrtW_(BD)-CrtZ_(BD). FIG. 11B illustrates pUC-pBAD-CrtI-m2-CrtY-CrtW_(BD). FIG. 11C illustrates pUC-pBAD-CrtI-m2-CrtY-CrtZ_(BD). FIG. 11D illustrates pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A)-idi.

FIG. 12 shows the results of HPLC analysis of C50-zeaxanthin, C50-canthaxanthin, and C50-astaxanthin produced in the case where E. coli was transformed with various plasmids containing CrtW and/or CrtZ derived from Brevundimonas sp. strain SD-212 and pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A) and cultured (Example 8). (a), (b), and (c) show the results of transformation with pUC-pBAD-CrtI-m2-CrtY-CrtZ_(BD), pUC-pBAD-CrtI-m2-CrtY-CrtW_(BD), and pUC-pBAD-CrtI_(N304P)-CrtY-CrtW_(BD)-CrtZ_(BD), respectively, together with pAC-CrtM_(F26A,W38A,F233S)-fds_(Y81A,V157A). Numerals 1, 2, and 3 in FIG. 12 represent the peaks of C50-zeaxanthin, C50-canthaxanthin, and C50-astaxanthin, respectively, and numerals with dashes (primes) represent cis peaks thereof.

FIG. 13 shows that idi co-expression remarkably increased the synthesis amount of C50-astaxanthin in E. coli transformed with pUC-pBAD-CrtI_(N304P)-CrtY-CrtW_(BD)-CrtZ_(BD) and pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A) (Example 9). (a) shows the results when co-expressing no idi, that is, the synthesis amounts of C50-carotene (n=3) and C50-astaxanthin in E. coli transformed with pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A) and pUC-pBAD-CrtI_(N304P)-CrtY-CrtW_(BD)-CrtZ_(BD). (b) shows the results when co-expressing idi, that is, the synthesis amounts of C50-carotene (n=3) and C50-astaxanthin in E. coli transformed with pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A)-idi and pUC-pBAD-CrtI_(N304P)-CrtY-CrtW_(BD)-CrtZ_(BD).

FIG. 14 shows the result of identification by NMR analysis, which indicates that a compound synthesized in E. coli transformed with pUC-pBAD-CrtI_(N304P)-CrtY-CrtW_(BD)-CrtZ_(BD) and pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A) was C50-astaxanthin (Example 10).

FIGS. 15A-15D illustrate plasmid maps used in Example 11. The plasmids contain, in addition to CrtW and/or CrtZ derived from Brevundimonas sp. strain SD-212, CrtG derived from Brevundimonas sp. strain SD-212 or CrtX derived from Pantoea ananatis. FIG. 15A illustrates pUC-pBAD-CrtI_(N304P)-CrtY-CrtG-CrtZ_(BD). FIG. 15B illustrates pUC-pBAD-CrtI_(N304P)-CrtY-CrtG-CrtW_(BD)-CrtZ_(BD). FIG. 15C illustrates pUC-pBAD-CrtI_(N304P)-CrtY-CrtX-CrtZ_(BD). FIG. 15D illustrates pUC-pBAD-CrtI_(N304P)-CrtY-CrtX-CrtW_(BD)-CrtZ_(BD).

FIG. 16 illustrates that more varieties of carotenoids can be produced by the additional expression of CrtG in carotenoid biosynthetic pathways (Example 11). Through the additional expression of CrtG, C₅₀-2-hydroxyastaxanthin and C₅₀-2,3,2′,3′-tetrahydroxy-β,β-carotene-4,4′-dione are produced from C₅₀-astaxanthin. C₅₀-caloxanthin and C₅₀-nostoxanthin are produced from C₅₀-zeaxanthin. Further, C₅₀-2-hydroxycanthaxanthin and C₅₀-2,2′-dihydroxycanthaxanthin are produced from C₅₀-canthaxanthin.

FIG. 17 illustrates that more varieties of carotenoids can be produced by the additional expression of CrtX in carotenoid biosynthetic pathways (Example 11). Through the additional expression of CrtX, C₅₀-astaxanthin-β-D-glucoside and C₅₀-astaxanthin-β-D-diglucoside are produced from C₅₀-astaxanthin. C₅₀-zeaxanthin-β-D-glucoside and C₅₀-zeaxanthin-3-D-diglucoside are produced from C₅₀-zeaxanthin.

FIG. 18 shows the results of confirmation of the fact that a great variety of carotenoids were able to be produced by the additional expression of CrtG or CrtX in E. coli strains that specifically synthesize C50-zeaxanthin and C50-astaxanthin, based on colony colors, cell pellet colors, and carotenoid extract colors (Example 11).

FIGS. 19A and 19B show the results of measurement of absorption spectra of carotenoid extracts obtained by the additional expression of CrtG or CrtX in E. coli strains that specifically synthesize C50-zeaxanthin and C50-astaxanthin (Example 11).

FIG. 20 show the results of HPLC analysis of a carotenoid produced by the additional expression of CrtG or CrtX in an E. coli strain that specifically synthesizes C50-zeaxanthin (Example 11). In FIG. 20, (b) shows that C50-caloxanthin (peak 3) and C50-nostoxanthin (peak 2) were produced by the additional expression of CrtG, and (c) shows that C50-zeaxanthin-β-D-diglucoside (peak 5) was produced by the additional expression of CrtX.

FIGS. 21A-21C illustrate plasmid maps used in Example 12. FIG. 21A illustrates pAC-hexPS. FIG. 21B illustrates pAC-FDS_(I78G,Y81A)-idi. FIG. 21C illustrates pUC-CrtM_(F26A,W38A,F233S).

FIG. 22 shows the results of biosynthesis of a C₅₅ carotenoid and a C₆₀ carotenoid (Example 12). When co-expressing CrtM_(F26A,W38A,F233S) with pAC-FDS_(I78G,Y81A)-idi, the C₅₅ carotenoid was produced in addition to C₄₅ and C₅₀ carotenoids. Further, when co-expressing CrtM_(F26A,W38A,F233S) with hexPS, the C₆₀ carotenoid was specifically synthesized. On the other hand, when using CrtM_(F26A,W38A), it was impossible to confirm the production of any of the C₅₅ carotenoid and the C₆₀ carotenoid.

FIG. 23 shows the results of measurement of carotenoid synthesis amounts when co-expressing AC-FDS_(I78G,Y81A)-idi or pAC-hexPS with 8 kinds of CrtM variants (pUC-CrtM variants) (Example 12). A larger amount of a C₅₅ carotenoid was synthesized in cells co-expressing FDS_(I78G,Y81A)-idi with CrtM_(F26A,F233S) or CrtM_(F26A,W38A,F233S). Further, a larger amount of a C₆₀ carotenoid was synthesized in cells co-expressing HexPS with CrtM_(W38A,F233S) or CrtM_(F26A,W38A,F233S).

DETAILED DESCRIPTION OF THE INVENTION

First, carotenoid biosynthetic pathways are described (see FIG. 1). Carotenoids are biosynthesized from mevalonic acid or pyruvic acid in nature. First, isopentenyl diphosphate (hereinafter referred to as “IPP”) and dimethylallyl diphosphate (hereinafter referred to as “DMAPP”), a compound obtained by the isomerization of IPP, are synthesized through a mevalonate pathway or a non-mevalonate pathway (MEP pathway in FIG. 1). Next, geranyl diphosphate (hereinafter referred to as “C₁₀PP”) is synthesized by condensation of IPP and DMAPP. Then, farnesyl diphosphate (hereinafter referred to as “C₁₅PP”) and geranylgeranyl diphosphate (hereinafter referred to as “C₂₀PP”) are synthesized by the sequential addition of two molecules of IPP. C₁₅PP is synthesized by a farnesyl diphosphate synthase, and C₂₀PP is synthesized by a geranylgeranyl diphosphate synthase.

In a pathway for synthesizing a carotenoid having 40 carbon atoms, phytoene is synthesized by condensation of two molecules of C₂₀PP with a phytoene synthase (CrtB), and serves as a precursor for a carotenoid (carotenoid backbone compound).

Phytofluene, ζ-carotene, neurosporene, lycopene, tetradehydrolycopene, and the like are synthesized by sequential desaturation of phytoene. Various carotenoids such as α-carotene, β-carotene, γ-carotene, δ-carotene, ε-carotene, lutein, zeaxanthin, canthaxanthin, fucoxanthin, astaxanthin, antheraxanthin, and violaxanthin are synthesized by modification of the ends of lycopene through cyclization or oxidation. It should be noted that carotenoids constructed only of carbon and hydrogen are classified as carotenes, while carotenoids containing an oxygen element in addition to carbon and hydrogen are classified as xanthophylls.

The present invention enables production of a carotenoid having 50 carbon atoms by alteration of biosynthetic pathways for carotenoids present in nature, and is directed to a production method for a carotenoid having 50 carbon atoms, wherein the production method comprises: culturing, in a medium, a cell transformed with a mutant phytoene desaturase gene; and obtaining a carotenoid having 50 carbon atoms from a culture after the culturing. It should be noted that, in this description, “50 carbon atoms” is sometimes simply referred to as “C₅₀”, and the same applies to, for example, 35, 40, 45, 55, and 60 carbon atoms.

In this description, the “carotenoid having 50 carbon atoms” is distinguished from a “carotenoid backbone compound of 50 carbon atoms” to be desaturated by a mutant phytoene desaturase, and refers to a compound whose number of double bonds has been increased by one or more by the desaturation of the carotenoid backbone compound of 50 carbon atoms. The carotenoid having 50 carbon atoms may undergo any modification, and also includes, for example, one having a β ring or an s ring at its ends, and one having a functional group containing an element other than carbon and hydrogen, such as a hydroxyl group or a keto group. Further, the carotenoid having 50 carbon atoms may be any carotenoid as long as the number of carbon atoms derived from a backbone compound is 50, and also includes, for example, one in which the total number of carbon atoms is 50 or more as a result of the addition of a functional group containing carbon, such as a methyl group or an acetyl group, by modification.

The “carotenoid backbone compound of 50 carbon atoms” is a precursor for the carotenoid having 50 carbon atoms, which may be desaturated by a mutant phytoene desaturase. Specific examples of the carotenoid backbone compound of 50 carbon atoms include: C₅₀-carotene (n=3) (16,16′-diisopentenylphytoene); and compounds in which the number of conjugated double bonds in C₅₀-carotene (n=3) is increased by 1 to 5. Specific examples of the compounds in which the number of the double bonds in C₅₀-carotene (n=3) is increased by 1 to 5 include: C₅₀-carotene (n=5), which is obtained by increasing the number of the double bonds by 1; C₅₀-carotene (n=7), which is obtained by increasing the number of the double bonds by 2; and C₅₀-ζ-carotene (n=11) and C₅₀-neurosporene (n=13) in FIG. 1. It should be noted that, in this description, “n”, which is described, for example, following common names for compounds, represents the number of conjugated double bonds.

In this description, the carotenoid backbone compound of 50 carbon atoms that has been desaturated by a mutant phytoene desaturase but has not undergone any modification afterwards is referred to as “desaturated carotenoid having 50 carbon atoms.” The desaturated carotenoid having 50 carbon atoms is a linear compound that does not have any functional group except for that derived from a backbone compound. The desaturated carotenoid having 50 carbon atoms is encompassed by the carotenoid having 50 carbon atoms. Specific examples of the desaturated carotenoid having 50 carbon atoms include compounds in which the number of double bonds in C₅₀-carotene (n=3) is increased by 1 to 6, and do not include C₅₀-carotene (n=3) itself. It should be noted that the compound in which the number of the double bonds in C₅₀-carotene (n=3) is increased by 6 is C₅₀-lycopene (n=15).

Further, in this description, the term “compound having a carotenoid backbone of 50 carbon atoms” is used in some cases as a concept including all of the carotenoid having 50 carbon atoms, the carotenoid backbone compound of 50 carbon atoms, the desaturated carotenoid having 50 carbon atoms, and the like.

The mutant phytoene desaturase that catalyzes a reaction for desaturating the carotenoid backbone compound of 50 carbon atoms is obtained by inducing a mutation in a wild-type phytoene desaturase (CrtI). The phytoene desaturase (CrtI) is an enzyme that desaturates phytoene having 40 carbon atoms. In nature, CrtI catalyzes a synthesis reaction of lycopene that contains 11 conjugated double bonds, by desaturating phytoene that contains 3 conjugated double bonds to sequentially introduce double bonds. The mutant phytoene desaturase in the present invention has an enhanced activity to desaturate the C₅₀ carotenoid backbone compound as compared to the wild-type phytoene desaturase by virtue of the introduction of a mutation.

The mutant phytoene desaturase in the present invention may be derived from any organisms including plants, bacteria, and the like as long as the mutant phytoene desaturase has an enhanced activity to desaturate the C₅₀ carotenoid backbone compound by virtue of a mutation. The mutant phytoene desaturase is preferably derived from microorganisms, more preferably derived from bacteria belonging to the genus Pantoea (formerly named the genus Erwinia), still more preferably derived from Pantoea ananatis (formerly named Erwinia uredovora). The amino acid sequence of the wild-type phytoene desaturase (CrtI) derived from Pantoea ananatis is set forth in SEQ ID NO: 1 of the sequence listing.

In the present invention, a mutant phytoene desaturase gene (crtI*) encodes the mutant phytoene desaturase. A mutation in the mutant phytoene desaturase gene may be any mutation as long as it achieves the object of the present invention. The mutation preferably causes a substitution of an amino acid corresponding to at least one amino acid selected from asparagine at position 304, phenylalanine at position 339, isoleucine at position 338, aspartic acid at position 395, and isoleucine at position 228 in the amino acid sequence set forth in SEQ ID NO: 1, more preferably causes at least a substitution of an amino acid corresponding to asparagine at position 304 in SEQ ID NO: 1 by proline or serine, still more preferably causes at least a substitution of an amino acid corresponding to asparagine at position 304 in SEQ ID NO: 1 by proline. Herein, the “amino acid corresponding to an amino acid at position X in SEQ ID NO: 1” defines that an amino acid as a target of the mutation is at position X counting from the N-terminus in SEQ ID NO: 1, but encompasses that the position X is expressed with a different numerical value in a phytoene desaturase having an amino acid sequence different from the amino acid sequence set forth in SEQ ID NO: 1.

The mutant phytoene desaturase gene is specifically exemplified by a phytoene desaturase gene derived from Pantoea ananatis (nucleotide sequence set forth in SEQ ID NO: 2 of the sequence listing), the nucleotide sequence having introduced therein a mutation causing a desired amino acid substitution. The mutant phytoene desaturase gene is exemplified by a gene having a nucleotide sequence of SEQ ID NO: 3 in which adenine (A) at position 911 is substituted by guanine (G) in the nucleotide sequence of SEQ ID NO: 2. The substitution of the nucleotide at position 911 causes a substitution of asparagine at position 304 in the amino acid sequence of SEQ ID NO: 1 by serine. It should be noted that SEQ ID NO: 2 has introduced therein non-synonymous mutations G1131A and A1476T. Further, the mutant phytoene desaturase gene is exemplified by a gene having a nucleotide sequence in which the nucleotide sequence AAC at positions 910 to 912 in the nucleotide sequence of SEQ ID NO: 2 is substituted by CCT, CCC, CCA, or CCG, more preferably CCT (nucleotide sequence set forth in SEQ ID NO: 28 of the sequence listing). The substitution of such bases causes a substitution of asparagine at position 304 in the amino acid sequence of SEQ ID NO: 1 by proline (amino acid sequence set forth in SEQ ID NO: 27 of the sequence listing). It should be noted that SEQ ID NO: 28 has introduced therein non-synonymous mutations G1131A and A1476T.

The nucleotide sequence of the mutant phytoene desaturase gene may be determined by producing a mutant gene library and screening a gene encoding an enzyme that has a function of interest from the library. The screening may be performed by, for example, a method described in Examples. Once the nucleotide sequence is determined, the mutant phytoene desaturase gene may be obtained by, for example, chemical synthesis, PCR using a cloned probe as a template, or a site-directed mutagenesis method.

The present invention includes the step of culturing, in a medium, a cell to be transformed with a mutant phytoene desaturase gene. The cell may be one originally harboring any other carotenoid biosynthetic gene, or may be one transformed with any other carotenoid biosynthetic gene. The other carotenoid biosynthetic gene is involved in a reaction upstream or downstream of a desaturating reaction for the C₅₀ carotenoid backbone compound. The upstream reaction corresponds to a pathway for supplying the C₅₀ carotenoid backbone compound, and the downstream reaction corresponds to a pathway for further modifying the C₅₀ desaturated carotenoid.

The pathway for supplying the C₅₀ carotenoid backbone compound is described. IPP, DMAPP, C₁₀PP, C₁₅PP, and C₂₀PP can be originally synthesized in many cells, in particular, all microorganisms. It is preferred that the cell in the present invention can synthesize geranylfarnesyl diphosphate (hereinafter sometimes referred to as “C₂₅PP”) from C₁₅PP and/or C₂₀PP, and can synthesize the C₅₀ carotenoid backbone compound via the condensation of two molecules of C₂₅PP. It is more preferred that the cell in the present invention be transformed with at least one of a gene encoding an enzyme that synthesizes C₂₅PP from C₁₅PP and/or C₂₀PP or a gene encoding an enzyme that synthesizes the C₅₀ carotenoid backbone compound via the condensation of two molecules of C₂₅PP.

The gene encoding the enzyme that synthesizes C₂₅PP from C₁₅PP and/or C₂₀22 may be any gene having a function of interest. Such gene is exemplified by a mutant gene of a farnesyl diphosphate synthase (FDS) derived from Geobacillus stearothermophillus, a moderate thermophilic bacterium belonging to the genus Geobacillus (Ohnuma, S. et al., J Biol Chem 271, 30748-30754 (1996), JP 2010-258989). The mutant gene is exemplified by a gene encoding a double mutant of FDS (FDS_(Y81A,V157A)) in which tyrosine at position 81 is substituted by alanine (Y81A) and valine at position 157 is substituted by alanine (V157A) (fds_(Y81A,V157A): SEQ ID NO: 4 of the sequence listing).

The gene encoding the enzyme that synthesizes the C₅₀ carotenoid backbone compound via the condensation of two molecules of C₂₅PP may be any gene having a function of interest. Such gene is exemplified by a mutant gene of a diapophytoene synthase (CrtM) derived from Staphylococcus aureus, which synthesizes a C₃₀ carotenoid backbone compound via the condensation of two molecules of C₁₅PP. The mutant gene is exemplified by a gene encoding a triple mutant of CrtM (CrtM_(F26A,W38A,F233S)) in which phenylalanine at position 26 and tryptophan at position 38 are substituted by alanine (F26A and W38A) and phenylalanine at position 233 is substituted by serine (F233S) CrtM_(F26A,W38A,F233S): SEQ ID NO: 5 of the sequence listing). It has been found that CrtM_(F26A,W38A,F233S) synthesizes the C₅₀ carotenoid backbone compound in an extremely efficient manner.

The cell of the present invention is preferably transformed with both of the gene encoding the enzyme that synthesizes C₂₅PP from C₁₅PP and/or C₂₀PP and the gene encoding the enzyme that synthesizes the C₅₀ carotenoid backbone compound from two molecules of C₂₅PP because the cell can produce the C₅₀ carotenoid backbone compound with high efficiency. In addition, the cell of the present invention may be transformed with a gene (idi) encoding an enzyme that isomerizes IPP into DMAPP (e.g., an isopentenyl diphosphate isomerase (Idi)) in addition to the above-mentioned genes. When the cell is transformed with the gene encoding the enzyme that isomerizes IPP into DMAPP, the production amount of the C₅₀ carotenoid backbone compound and the specificity can be further improved.

The pathway for further modifying the C₅₀ desaturated carotenoid is described. The C₅₀ desaturated carotenoid is considered to correspond to lycopene or tetradehydrolycopene in nature, and it is predicted that the C₅₀ desaturated carotenoid may be modified by various enzymes involved in the modification of lycopene. The cell in the present invention may contain a gene encoding an enzyme that cyclizes the ends of the C₅₀ desaturated carotenoid and/or a gene encoding an enzyme that oxidizes the C₅₀ desaturated carotenoid by oxygenation. In addition, when the cell in the present invention has the gene encoding the enzyme that cyclizes the ends of the C₅₀ desaturated carotenoid (in particular, β-cyclization), the cell may be transformed with a gene encoding an enzyme that hydroxylates a cyclic moiety (in particular, a β-ring) and/or a gene encoding an enzyme that ketolates a cyclic moiety (in particular, a β-ring). It should be noted that the β-ring has the same meaning as a β-ionone ring.

The gene encoding the enzyme that cyclizes the ends of the C₅₀ desaturated carotenoid may be any gene having a function of interest. Such gene is exemplified by a gene (crtY) encoding a lycopene cyclase (CrtY) derived from Pantoea ananatis, which synthesizes β-carotene from lycopene (Misawa N. et al., J Bacteriol 172, 6704-6712 (1990)).

In a carotenoid having β-cyclized ends, a gene encoding an enzyme that hydroxylates the β-ring and/or a gene encoding an enzyme that ketolates the β-ring may be any gene having a function of interest. Such gene is exemplified by a β-ionone ring-3-hydroxylase (CrtZ) gene (crtZ) or β-ionone ring-4-ketolase (β-ionone ring-4-oxygenase) (CrtW) gene (crtW) derived from a marine bacterium Paracoccus sp. strain N81106 (formerly named Agrobacterium aurantiacum) or derived from a marine bacterium Brevundimonas sp. strain SD-212 (Misawa N. et al., J Bacterial 177, 6575-6584 (1995); Nishida, Y. et al., Appl Environ Microbiol 71, 4286-4296 (2005)). A nucleotide sequence in which crtZ and crtW derived from Paracoccus sp. strain N81106 and crtY described above are combined is set forth in SEQ ID NO: 6. A β-ionone ring-3-hydroxylase catalyzes a reaction for hydroxylating carbon at the 3-position of a β-ring, and a β-ionone ring-4-ketolase catalyzes a reaction for oxygenating carbon at the 4-position of a β-ring to form a carbonyl group (keto group).

The gene encoding the enzyme that oxidizes the C₅₀ desaturated carotenoid may be any gene having a function of interest. Such gene is exemplified by a spheroidene monooxygenase (CrtA) gene (crtA) derived from Rhodobacter sphearoides (SEQ ID NO: 7). A spheroidene monooxygenase is an enzyme that catalyzes an oxidation reaction for converting spheroidene into spheroidenone by inserting an oxygen atom.

The cell of the present invention may be transformed with genes encoding various enzymes involved in the pathway for modifying the C₅₀ desaturated carotenoid other than those described above, depending on the kind of a carotenoid to be produced. For example, when the cell is transformed with a β-ionone ring-2-hydroxylase (CrtG) gene (expressed as “CrtV” in WO 2005/049643 A1) derived from Brevundimonas sp. strain SD-212 (Nishida, Y. et al, Appl Environ Microbiol 71, 4286-4296, 2005), a C₅₀ carotenoid having a β-ring hydroxylated at the 2-position and the 2′-position, the organic synthesis of which has been considered to be difficult, can also be produced. Further, when the cell is transformed with a zeaxanthin glucosyl transferase (crtX) gene derived from Pantoea ananatis (Misawa N, J Bacteriol 172, 6704-6712, 1990), a C₅₀ carotenoid in which hydroxyl groups at the 3-position and 3′-position of a β-ring are glycosidized can also be produced.

A cell capable of producing a C₅₀ carotenoid in the present invention may be produced by selecting an appropriate expression vector and employing a known foreign gene introduction and expression method (e.g., Sambrook, J., Russel, D. W., Molecular Cloning A Laboratory Manual, 3rd Edition, CSHL Press, 2001). The cell is obtained by: preparing a gene to be introduced into the cell by transformation by using a conventional method such as a PCR method; incorporating the gene into an expression vector suitable for a host by using a conventional method; selecting a vector of interest; and transforming a host cell with the vector by using a conventional method. When transforming a cell with two or more kinds of genes, the cell may be transformed with the plurality of genes incorporated into the same expression vector, or may be cotransformed with the plurality of genes incorporated into different expression vectors.

The cell serving as the host is not limited, but microorganisms such as E. coli, Bacillus subtilis, and yeast are preferred in view of shortening of a culturing time and ease of cloning. In particular, E. coli and yeast are preferred. Suitable examples of the E. coli include: cloning strains such as Escherichia coli XL1-Blue (hereinafter simply referred to as “E. coli XL1-Blue”); expression strains such as HB101 and BL21; and gene knockout strains in which the synthesis amount of a terpene precursor is large, such as JW1750 ΔgdhA (glutamate dehydrogenase-deficient) and JW0110 ΔaceE (pyruvate dehydrogenase-deficient) (Baba, T. et al.; Mol Syst Biol 2, 2006 0008 (2006)). Suitable examples of the yeast include standard budding yeast INVSc1 (invitrogen) and YPH499 (stratagene).

The expression vector into which the gene is applied is not particularly limited and may be a vector to be generally used. For example, when the host is E. coli, there are given ones derived from pUC18, pACYC184, and the like. When the host is Bacillus subtilis, there are given pUB110, pE194, pC194, pHY300PLK DNA, and the like. And when the host is yeast, there are given pRS303, YEp213, TOp2609, and the like.

Whether or not a gene of interest is introduced into the host cell may be confirmed by a conventional method such as a PCR method, a Southern hybridization method, or a northern hybridization method.

The production method for a C₅₀ carotenoid of the present invention includes the step of culturing, in a medium, the cells as the transformants obtained as described above. The medium may be any medium containing a substance that may serve as a supply source for the C₅₀ carotenoid backbone compound, and may be any medium containing an ingredient to be generally used for cell culturing. For cells in which the C₅₀ carotenoid backbone compound is synthesized by the metabolism of IPP and DMAPP, the medium may be any medium containing a carbon source that may serve as a supply source for IPP and DMAPP. Examples of such carbon source include a variety of sugars such as glucose.

A temperature at the time of the culturing is not particularly limited but is set to preferably 18 to 30° C., more preferably 20 to 30° C.

Culture period is also not particularly limited but the culturing is performed for preferably 12 to 72 hours, more preferably 24 to 48 hours after the expression of the gene introduced by the transformation.

The C₅₀ carotenoid can be collected from the culture after the culturing in accordance with a method to be generally employed for obtaining a product such as a carotenoid from cells of microorganisms or the like. It may also be possible to separate only the cells from the culture and obtain the carotenoid from the cells.

It should be noted that the present invention is also directed to a mutant phytoene desaturase gene, a mutant phytoene desaturase encoded by the mutant phytoene desaturase gene, and a cell capable of desaturating a C₅₀ carotenoid backbone compound to produce a C₅₀ carotenoid, which is transformed with the mutant phytoene desaturase gene.

Further, the present invention can be utilized in the highly efficient synthesis of a desaturated C₅₅ carotenoid, a desaturated C₆₀ carotenoid, or the like. For example, the desaturated C₅₅ carotenoid as well as the C50 carotenoid can be produced with high efficiency by utilizing the present invention using a double mutant of a farnesyl diphosphate synthase (FDS) (FDS_(I78G,Y81A)) in which isoleucine at position 78 is substituted by glycine (I78G) and tyrosine at position 81 is substituted by alanine (Y81A) in FDS (Ohnuma S et al., J Biol Chem 273, 26705-26713, 1998). Further, the desaturated C₆₀ carotenoid can be produced with high efficiency by utilizing the present invention using a C₃₀PP synthase (HexPS) derived from Micrococcus luteus (Shimizu N et al, J Bacteriol 180, 1578-1581, 1998), that is, by co-expressing FDS_(I78G,Y81A) or HexPS with the CrtM variant.

EXAMPLES

Hereinafter, the present invention is specifically described by way of Examples. However, the present invention is by no means limited thereto.

(Reference Example 1) Synthesis of C₅₀ Carotenoid Backbone Compound

(1) Supply of C₅₀ Carotenoid Raw Material, C₂₅PP

A mutant gene fds_(Y81A, V157A) was used in order to efficiently synthesize C₂₅PP in E. coli. fds_(Y81A, V157A) is a mutant of a farnesyl diphosphate synthase (FDS) derived from Geobacillus stearothermophillus. The mutation Y81A is derived from Ohnuma, S. et al., J Biol Chem 271, 30748-30754 (1996). Further, the inventors of the present invention produced fds_(Y81A,V157A) encoding a mutant enzyme having further shifted size specificity for a substrate by further introducing a mutation into a mutant gene fds_(Y81A) through the use of the screening method for a terpene synthase gene disclosed in JP 2010-258989.

(2) Synthesis of C₅₀ Carotenoid Backbone Compound

A mutant gene crtM_(F26A,W38A,233S) was used for the synthesis of a C₅₀ carotenoid backbone compound (16,16′-diisopentenylphytoene) via the condensation of two molecules of C₂₅PP. A diapophytoene synthase (CrtM) derived from Staphylococcus aureus is originally an enzyme that synthesizes a C₃₀ backbone carotenoid via the condensation of two molecules of C₁₅PP. The inventors of the present invention introduced a mutation into a crtM gene in order to produce an enzyme having improved size selectivity for a substrate (Umeno et al., J Bacteriol 184, 6690-6699 (2002), Umeno et al., Nucleic Acids Res 31, e91 (2003)). It was found that a C₅₀ carotenoid backbone compound was synthesized in a trace amount by feeding C₂₅PP to a double mutant of CrtM having mutations F26A and W38A (Non Patent Literature 7).

The inventors also found that a triple mutant of CrtM having introduced therein mutations F26A, W38A, and F233S synthesized a C₅₀ carotenoid backbone compound from two molecules of C₂₅PP in an extremely efficient manner, and synthesized a C₆₀ carotenoid backbone compound, though in a trace amount, when C₃₀PP was fed (Maiko Furubayashi, Mayu Ikezumi, Kyoichi Saito, Daisuke Umeno. Activity evolution of unnatural carotenoid synthetic pathways, Annual Meeting of the Kanto Branch of the Japan Society for Bioscience, Biotechnology, and Agrochemistry, 2010, Oct. 9, 2010: Daisuke Umeno, Maiko Furubayashi, Mayu Ikezumi, Akinori Katabami, Ling Li, Jun Kajiwara. Creation and development of unnatural biosynthetic pathways, The Third Annual Meeting of the Japanese Society for Cell Synthesis Research, Institute of Industrial Science, the University of Tokyo, Nov. 12, 2010: Furubayashi M, Saito K, Umeno D, In-laboratory genetic drift of carotenoid synthase and its evolution of size specificity. The international chemical congress of Pacific basin societies, Hawaii, USA, Dec. 17, 2010: Maiko Furubayashi, Mayu Ikezumi, Kyoichi Saito, Daisuke Umeno. Selective synthesis of unnatural carotenoid by combinatorial expression of enzyme mutant, Annual Meeting of the Japan Society for Bioscience, Biotechnology, and Agrochemistry, 2011, March 2011). It was found that C₅₀-carotene (n=3), a C₅₀ carotenoid backbone compound, was efficiently (about 61%) produced by co-expressing fds_(Y81A, V157A) for supplying C₂₅PP and crtM_(F26A,W38A,F233S) in E. coli XL1-Blue.

(3) It should be noted that the transformation of E. coli XL1-Blue with fds_(Y81A, V157A) and crtM_(F26A,W38A,F233S) was performed by producing plasmids pAC-crtM_(F26A,W38A,F233S) (FIG. 2G: SEQ ID NO: 8) and pUC-fds_(Y81A,V157A) (FIG. 2F: SEQ ID NO: 9) in accordance with a conventional method.

pAC-crtM_(F26A,W38A,F233S) was produced by inserting lac promoter/operator (lacPO)-crtM_(F26A,W38A,F233S) into the BamHI site of a pACmod vector (Claudia Schmidt-Dannert et al., Nat. Biotechnol., 18: 750-753 (2000)).

pUC-fds_(Y81A,V157A) was produced by inserting an fds_(Y81A,V157A) gene downstream of lacPO of a pUC18 Nm vector (Umeno D. et al, J Bacteriol 184, 6690-6699 (2002)).

The transformed E. coli was cultured in accordance with the technique disclosed in Non Patent Literature 7.

The analysis of the produced carotenoid was performed by HPLC also in accordance with the method disclosed in Non Patent Literature 7.

(Example 1) Improved Synthesis of C₅₀ Carotenoid Backbone Compound

In addition to fds_(Y81A,V157A) and CrtM_(F26A,W38A,F233S), a gene (derived from E. coli genome) encoding an isopentenyl diphosphate isomerase (Idi) was expressed in E. coli XL1-Blue, and the E. coli was cultured. The transformation of E. coli XL1-Blue with fds_(Y81A, V157A) and crtM_(F26A,W38A,F233S) was performed by producing plasmids pAC-crtM_(F26A,W38A,F233S)-idi (FIG. 2H: SEQ ID NO: 10) and pUC-fds_(Y81A,V157A) (FIG. 2F: SEQ ID NO: 9) in accordance with a conventional method. pAC-crt_(MF26A,W38A,F233S)-idi was produced by inserting lacPO-idi upstream of the ClaI site of pAC-crt_(MF26A,W38A,F233S). pUC-fds_(Y81A,V157A) was produced in the same manner as in Reference Example 1. The culturing of the transformed E. coli and the analysis of the produced carotenoid were performed in the same manner as in Reference Example 1.

FIG. 3 shows the results. The production amount of C₅₀-carotene (n=3), a C₅₀ carotenoid backbone compound, was increased in the case of introducing Idi as compared to the case of not introducing Idi. Further, phytoene, a C₄₀ carotenoid backbone compound, was synthesized in a large amount in the case of not introducing Idi, whereas the production amount of phytoene was decreased and the specificity of the production of C₅₀-carotene (n=3) was improved in the case of introducing Idi. The synthetic efficiency of C₅₀-carotene (n=3) was found to be improved (about 90%) in the case of introducing Idi.

(Reference Example 2) Desaturation of C₅₀ Carotenoid Backbone Compound by Wild-Type CrtI

The inventors of the present invention found that a carotenoid having a C₅₀ carotenoid backbone compound was desaturated by CrtI (phytoene desaturase encoded by a gene having a nucleotide sequence of SEQ ID NO: 2 (having introduced therein non-synonymous mutations G1131A and A1476T)) (Non Patent Literature 9). However, it was found that its production amount is extremely small and only 25% of the C₅₀ carotenoid backbone compound was desaturated (Non Patent Literature 8).

Meanwhile, the inventors of the present invention found that the constitutive expression of wild-type CrtI using a lac promoter (lacP) or the like remarkably inhibited cell growth, and remarkably destabilized cell pigmentation. However, no clear cytotoxicity was observed in cells having a pathway to the synthesis/accumulation of the C₅₀ carotenoid backbone compound (e.g., the cells obtained in Reference Example 1 and Example 1).

The inventors of the present invention attempted to ligate crtI downstream of an araBAD promoter capable of reducing leaky expression (low-level expression occurring without any induction) and express the ligation product in E. coli XL1-Blue. A group of carotenoid biosynthetic genes upstream of crtI were constitutively expressed by lacP. After the density/number of the cells had reached sufficient levels, it was attempted to make pigment in the cells by inducing the expression of crtI to desaturate the C₅₀ carotenoid backbone compound.

The plasmid used is pUC-pBAD-crtI (having crtI introduced into the crtI variant part in FIG. 2B). The lac promoter/operator (lacPO) of a pUC18 Nm vector (Umeno D. et al, J Bacteriol 184, 6690-6699 (2002)) was removed, and an araC gene/araBAD promoter sequence derived from a pBADHisA vector (invitrogen) was inserted. crtI was inserted into the XhoI-ApaI site downstream of the promoter. crtI is a gene encoding a wild-type phytoene desaturase derived from Pantoea ananatis (Misawa N. et al., J Bacteriol 172, 6704-6712 (1990)).

The problem of the cytotoxicity was overcome in E. coli transformed with pUC-pBAD-crtI. However, the C₅₀ carotenoid backbone compound was desaturated with extremely low efficiency, which was almost unrecognizable.

(Example 2) Acquisition of crtI Variant Desaturating C₅₀ Carotenoid Backbone Compound

Based on the evolutionary engineering of a phytoene desaturase (CrtI), it was attempted to acquire a crtI variant that desaturates more efficiently the C₅₀ carotenoid backbone compound. FIG. 4A illustrates an overview of procedures of this example, and FIG. 4B illustrates a screening principle.

First, a plasmid pUC-pBAD-crtI containing wild-type crtI (represented by “pUC-I” in FIG. 4B) was used as a template, and a random mutation was introduced into crtI by an error-prone PCR method using MnCl₂ (Cadwell et al.: PCR Methods Appl 2, 28-33 (1992)) The obtained PCR product was digested with restriction enzymes XhoI and ApaI, and ligated into the restriction enzyme sites of XhoI and ApaI downstream of an araBAD promoter in a pUC-pBAD vector. E. coli XL10-Gold (Stragatene) was transformed with the ligation product, and part thereof was spread on a Luria Bertani (LB) solid medium. The remaining transformed E. coli was added to 10 mL of an LB liquid medium and cultured at 37° C. overnight. After that, a plasmid was extracted from 2 mL of the culture and used as a “crtI variant plasmid library.” Further, the number of the transformed cells (library size), which was calculated from the number of the colonies formed on the solid medium, was 10⁵ cfu/transformation.

E. coli XL1-Blue harboring a plasmid pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A) (represented by “pAC-C₅₀” in FIG. 4B: SEQ ID NO: 11) was transformed with the thus obtained plasmid library and spread on an LB solid medium (containing 50 μg/mL carbenicillin and 30 μg/mL chloramphenicol) on which a nitrocellulose filter was mounted. The cells were cultured at 37° C. for 24 hours until the colonies were formed, and then left to stand still at room temperature for an additional 48 hours.

As a control, E. coli XL1-Blue was transformed with pUC-pBAD-crtI containing wild-type crtI. In this case, the colonies developed a flesh color. In the group of about 2,000 colonies obtained by transformation with the crtI variant plasmid library, 8 colonies developing a particularly intense reddish violet color were visually detected. The colonies were named CrtI-m1, m2, and m8, respectively. As illustrated in FIG. 4B, C₅₀-lycopene (n=15), a C₅₀ desaturated carotenoid, is a pigment developing a reddish violet color, and it is estimated that, as a result of the efficient synthesis of such compound, the colonies developed a reddish violet color.

The bacterial strain of each of the colonies was cultured in an LB liquid medium at 37° C. for 12 hours to extract a plasmid, and the nucleotide sequence of a crtI variant contained in the plasmid was analyzed by a dideoxy method. Table 1 below shows the analysis results.

TABLE 1 Analysis results of gene mutations in CrtI variants Gene mutations (amino acid mutations are Sample names shown in parentheses) CrtI-m1 A195G, A594G, T1016C (F339S) CrtI-m2 A911G (N304S) CrtI-m3 None CrtI-m4 T1015C (F339L), G1183A (D395N) CrtI-m5 None CrtI-m6 A682G (I228V), A1012G (I338V) CrtI-m7 T144C, A1012G (I338V) CrtI-m8 A911G (N304S), T1017C

It was found that CrtI-m2 and CrtI-m8 had the same mutation in which asparagine (N) at position 304 was substituted by serine (S) (N304S). Further, CrtI-m1 and CrtI-m4 each had a mutation in which phenylalanine (F) at position 339 was substituted by serine (S) or leucine (L) (F339S or F339L), and CrtI-m6 and CrtI-m7 each had a mutation in which isoleucine (I) at position 338 was substituted by valine (V) (I338V). It was estimated that the CrtI variants each had an enhanced ability to desaturate a C₅₀ carotenoid backbone compound by virtue of those mutations.

(Example 3) Synthesis of Desaturated C₅₀ Carotenoid by CrtI Variant

Of the colonies obtained in Example 2, each of CrtI-m1, m2, m4, m6, and m8, which showed enhanced pigmentation as compared to wild-type CrtI, was confirmed for its ability to desaturate a C₅₀ carotenoid backbone compound.

E. coli XL1-Blue harboring pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A) was transformed with each of the plasmids pUC-pBAD-CrtI-m1, m2, m4, m6, and m8. The transformant was spread on an LB agar medium on which a nitrocellulose (NC) membrane was contracted, and cultured at 37° C. for 24 hours. The plasmids pUC-pBAD-CrtI-m1, m2, m4, m6, and m8 have various mutant genes mut1, mut2, mut4, mut6, and mut8 inserted into the crtI* part in pUC-pBAD-crtI*-crtY of FIG. 2C, respectively, and the nucleotide sequence of the plasmid having inserted therein mut2 is set forth in SEQ ID NO: 12.

After the formation of the colonies, the NC membrane with the colonies was transferred onto an LB agar medium containing 0.2% arabinose, followed by incubation at room temperature. The colonies were inoculated into 2 mL of an LB medium (containing 50 μg/mL carbenicillin and 30 μg/mL chloramphenicol) and cultured at 37° C. overnight. 300 μL of the culture medium were inoculated into 30 mL of a TB medium (containing 50 μg/mL carbenicillin and 30 μg/mL chloramphenicol), followed by incubation under shaking at 30° C. and 200 rpm. After the incubation under shaking for 24 hours, 20% arabinose was added to the culture medium so as to have a final concentration of 0.2%, followed by incubation under shaking (30° C.) for an additional 48 hours.

The culture medium was measured for its OD₆₀₀, and the cells were harvested by centrifugation. The pellet obtained by harvesting the cells was washed with physiological saline, and a lipid-soluble fraction was extracted with 10 mL of acetone. A 300-μL aliquot was fractionated from the extract and measured for its absorbance spectrum with Spectra Max 384 (Molecular Device).

FIGS. 5A and 5B show the results. FIG. 5A is a photograph showing the actual color of each acetone extract. The absorbance of the acetone extract at around 500 to 600 nm was increased in E. coli expressing mut2, mut8, mut1, mut4, or mut6 as compared to E. coli expressing wild-type crtI (WT) (FIG. 5B). In particular, CrtI-m2 and CrtI-m8 having crtI variants with N304S exhibited intense colors and absorbances. In the case of expressing those CrtI variants, a peak (shoulder) on the rightmost side is located at about 540 nm, indicating that a six-step desaturated product (15 conjugated double bonds) is contained in a large amount. The synthesis amount of the C₅₀ desaturated carotenoid was the largest in the case of introducing CrtI-m2 or CrtI-m8.

Next, the carotenoid synthesized in the case of CrtI-m2 was subjected to HPLC analysis. 1 mL of hexane and 35 mL of 10% NaCl were added to the acetone extract to extract a carotenoid fraction into a hexane phase. 75% of the hexane extract were collected and dehydrated with addition of a small amount of MgSO₄. The hexane solvent was removed by nitrogen, and the carotenoid fraction was finally concentrated into 100 μL of hexane. Thus, a carotenoid extract was obtained. 25 μL (75%×25%=19% of the total volume, corresponding to 7.3 mL of the medium) of the resultant carotenoid extract were injected into an HPLC-photodiodearray system. HPLC analysis was performed in accordance with the conditions of Takaichi, S. Photosynth Res, 65, 93-99 (2000) (column: Waters Spherisorb™ 5.0 μm ODS2 4.6 mm×250 mm Column, eluent: acetonitrile/tetrahydrofuran/metanol (58:7:35) 2 mL/min, detector: photodiode array (190 to 800 nm)). Further, the mass spectrometry of a sample fractionated by HPLC was performed through the use of an M-2500 Hitachi double-focusing mass spectrometer (Hitachi, Ltd.) at a field desorption mode (Takaichi (1993) Org. Mass Spectrom. 28: 785-788)).

As a result, it was found that, in CrtI-m2, a compound (C₅₀-lycopene) obtained by the six-step desaturation of the C₅₀ carotenoid backbone compound was synthesized in a large amount (peak 3 in FIG. 6B). On the other hand, it was found that, in the case of transformation with wild-type CrtI, almost no desaturated compound was synthesized (FIG. 6A).

The analysis results for peak 2 and peak 3 in FIG. 6B are shown below. C₅₀-carotene (n=3): A sample corresponding to peak 2 in FIG. 6B was fractionated by HPLC and subjected to mass spectrometry. The sample showed a much longer HPLC elution time than that of a desaturated carotenoid having the same size, and hence was estimated to be a compound having a C₅₀ carotenoid backbone. The absorption spectrum was similar to that of phytoene (n=3). The mass number obtained by the mass spectrometry was 680, which was consistent with that of a putative structure (Compound No. 2 in FIG. 1).

C₅₀-lycopene (n=15): A sample corresponding to peak 3 in FIG. 6B was fractionated by HPLC and subjected to mass spectrometry. The HPLC elution time was relatively short. The absorption spectrum was similar to the literature value of 3,4,3′,4′-tetradehydrolycopene (n=15) (Karrer and Rutschmann (1945) Helv. Chim. Acta 28: 793-795), and the absorption spectrum had the feature of an acyclic carotenoid. The mass number obtained by the mass spectrometry was 668, which was consistent with that of a putative structure (Compound No. 3 in FIG. 1).

(Example 4) Cyclization of Desaturated C₅₀ Carotenoid

C₅₀-lycopene (n=15), a desaturated C₅₀ carotenoid, was cyclized by using a crtY gene encoding a lycopene cyclase, which synthesizes a β-carotenoid from lycopene.

A plasmid pUC-pBAD-crtI/CrtI-m2-crtY was used for transformation with the crtY gene. The plasmid was produced by inserting an SpeI site following an ApaI site in pUC-pBAD-crtI/CrtI-m2 and inserting the crtY gene into the ApaI/SpeI site. The crtY gene is a gene encoding a lycopene cyclase derived from Pantoea ananatis (Misawa N. et al., J Bacteriol 172, 6704-6712 (1990)). It should be noted that the expression “CrtI-m2” in the plasmid means that a mutant gene crtI_(N304S) derived from CrtI-m2 has been inserted. The nucleotide sequence of pUC-pBAD-CrtI-m2-crtY is set forth in SEQ ID NO: 13.

E. coli XL1-Blue harboring pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A) was transformed with pUC-CrtI-m2-CrtY. As a control, transformation was performed with crtY together with wild-type crtI in place of crtI-m2. The E. coli was spread on an LB agar medium on which a nitrocellulose (NC) membrane was mounted, and cultured at 37° C. for 24 hours. After the formation of colonies, the NC membrane with the colonies was transferred onto an LB agar medium containing 0.2% arabinose, followed by incubation at room temperature. Then, the color of the colonies was observed.

FIG. 7 shows the photographs of the transformed E. coli cultured for 0 to 48 hours. The E. coli transformed with a plasmid having crtY downstream of a crtI variant derived from CrtI-m2 (“CrtI₂Y” in FIG. 7) developed a very deep red color.

Further, the acetone extract was subjected to HPLC analysis and mass spectrometry by the same techniques as those of Example 3.

A carotenoid having a maximum absorption peak at 502 nm was detected by the HPLC analysis (peak 7 in FIG. 6D). This peak was confirmed in only a trace amount in the case of wild-type CrtI (peak 5 in FIG. 6C). FIG. 1 illustrates the putative structure of the thus obtained C₅₀ carotenoid and a synthetic pathway therefor. It should be noted that the numbers of the respective compounds in FIG. 1 correspond to numbers in the chromatograms of FIGS. 6A-6I.

C₅₀-β-carotene: A sample corresponding to peak 7 in FIG. 6D was fractionated by HPLC and subjected to mass spectrometry. The HPLC elution time was longer than that of C₅₀-lycopene. It is known that a similar behavior is shown in a C₄₀ carotenoid as well. The absorption spectrum was almost consistent with that of a chemically synthesized product (Khachik and Beecher (1985) J. Chromatogr. 346: 237-246) (FIG. 9C). The absorption spectrum was similar to one having the feature of a bicyclic carotenoid. The mass number obtained by mass spectrometry was 668, which was consistent with that of a putative structure (Compound No. 7 in FIG. 1).

(Example 5) Extension of Cyclic C₅₀ Carotenoid Pathway by Additional Modification

An oxocarotenoid was synthesized by further extending the pathway for synthesizing a cyclic C₅₀ carotenoid confirmed in Example 4.

pUC-pBAD-crtI-crtWZY (FIG. 2D: SEQ ID NO: 14), a plasmid containing crtW, crtZ, and crtY, was used. As for the plasmid, crtW, crtZ, and crtY of Paracoccus sp. strain N81106 (SEQ ID NO: 6) were amplified from a plasmid pAK32 (Misawa N. et al., J Bacteriol 177, 6575-6584 (1995)) by PCR. Through the use of crtW, crtZ, and crtY thus obtained, pUC-pBAD-crtI-crtWZY and pUC-pBAD-CrtI-m2-crtWZY were constructed. In the same manner as the technique of Example 4, the produced plasmids were introduced into E. coli (XL1-Blue) together with pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A), and the E. coli was cultured, followed by observing the color of colonies.

As a result, in a system having introduced therein pUC-pBAD-CrtI-m2-crtWZY, the colonies developed a vivid red color (the second line from the right in FIG. 8C: CrtWZY in CrtI*).

The spectrum of the acetone extract was measured in the same manner as in Example 4. As a result, in the case of transformation with pUC-pBAD-CrtI-m2-crtWZY, an intense peak appeared at 508 nm (CrtI*-CrtWZY in FIG. 8A). On the other hand, in the case of using wild-type CrtI, only a trace amount of a pigment was accumulated (CrtI-CrtWZY in FIG. 8A). HPLC analysis was performed in the same manner as in Example 4. As a result, a novel carotenoid having high polarity and having a maximum absorption peak at 502 nm was obtained as a major peak (peak 9 in FIG. 6F). FIG. 1 illustrates the putative structure of the thus obtained carotenoid and a synthetic pathway therefor. It should be noted that the numbers of the respective compounds in FIG. 1 correspond to numbers in the chromatograms of FIGS. 5A and 5B.

(Example 6) Oxidation of Desaturated C₅₀ Carotenoid

The total synthesis of crtA was performed by optimizing a codon for E. coli based on the amino acid sequence of CrtA derived from Rhodobacter sphearoides (SEQ ID NO: 7, commissioned to DNA2.0). Plasmids pUC-pBAD-crtI-crtA and pUC-pBAD-CrtI-m2-crtA were produced by using the synthesized gene (FIG. 2E: SEQ ID NO: 15).

By the same techniques as those of Example 5, E. coli was transformed with the produced plasmids, and the E. coli was cultured, followed by observing the color of colonies and analyzing the spectrum of the acetone extract.

As a result, in the case of transformation with pUC-pBAD-CrtI-m2-crtA, the colonies developed a vivid color (the first line from the right in FIG. 8C: CrtA in CrtI*). Further, the acetone extract gave a broad absorption spectrum peculiar to an oxocarotenoid (CrtI*-CrtA in FIG. 8B). In the case of co-expression of CrtA with wild-type CrtI, the colonies developed only a tint color. HPLC analysis was performed in the same manner as in Example 5. As a result, a novel carotenoid having high polarity appeared as a major peak (peak 11 in FIG. 6H).

(Example 7) Acquisition of crtI Variant Having High Ability to Desaturate C₅₀ Carotenoid Backbone Compound

The biosynthetic pathways for C50-lycopene and C50-β-carotene realized in Examples 3 and 4 left something to be improved in efficiency, because the C50 backbone (C50-carotene (n=3)) still remained. Thus, the acquisition of a CrtI variant having a higher ability to desaturate the C50 backbone was attempted. Of the CrtI variants obtained in Example 2, CrtI-m2 showed the highest C50 backbone desaturation efficiency. Thus, with attention focused on the amino acid substitution N304S of CrtI-m2, the site-directed total substitution of an amino acid at position 304 was performed to search a variant having higher desaturation efficiency.

First, a plasmid pUC-pBAD-crtI containing wild-type CrtI was used as a template, and a library was prepared by PCR using a primer having a randomized amino acid at position 304 (NNK codon) and the subsequent cloning. The resultant plasmid library was screened by the same method as that described in Example 2. Colonies developing a redder color than those of wild-type CrtI were searched to isolate plasmids of CrtI variants. Those CrtI variants were screened again. As a result, products substituted by glycine (G), serine (S), asparagine (N), proline (P), and alanine (A) gave particularly red colonies. Of those, a product substituted by P (codon: CCT) gave the reddest colonies. The amino acid sequence of CrtI having the product substituted by P is set forth in SEQ ID NO: 27 of the sequence listing, and a nucleotide sequence encoding the amino acid sequence is set forth in SEQ ID NO: 28.

Next, the synthesis amounts of C50-lycopene by CrtI-m2 and CrtI_(N304P) were analyzed by HPLC. E. coli XL1-Blue was transformed with pUC-pBAD-crtI-m2 or pUC-pBAD-crtI_(N304P) together with pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A). After the formation of colonies, an NC membrane with the colonies was transferred onto an LB agar medium containing 0.2% arabinose, followed by incubation at room temperature. The colonies were inoculated into 2 mL of an LB medium (containing 50 μg/mL carbenicillin and 30 μg/mL chrolamphenicol) and cultured at 37° C. overnight. 300 μL of the culture medium were inoculated into 30 mL of a TB medium (containing 50 μg/mL carbenicillin and 30 μg/mL chrolamphenicol), followed by incubation under shaking at 30° C. and 200 rpm. After the incubation under shaking for 36 hours, 20% arabinose was added to the culture medium so as to have a final concentration of 0.20, followed by incubation under shaking (30° C.) for an additional 36 hours. HPLC analysis was performed by the method shown in Example 3.

FIG. 10 shows the results. In the case of using CrtI_(N304P), the synthesis amount of C50-lycopene accumulated in the cells showed substantially no increase, whereas the production amount of C50-carotene (n=3) showed a clear decrease as compared to CrtIm2. This is probably because most of C50-lycopene undergoes decomposition because of its low intracellular stability, although the biosynthesis amount of C50-lycopene (desaturation efficiency of C50-carotene) is large (high).

Next, whether or not C50-β-carotene was increased more by using CrtI_(N304P) was investigated. First, a pUC-pBAD-crtI_(N304P)-crtY plasmid was prepared. E. coli XL1-Blue was transformed with the plasmid and pUC-pBAD-crtI-m2-crtY together with pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A), and culturing, extraction, and HPLC analysis were performed in the same manner as in the above-mentioned sections.

As a result, C50-carotene (n=3) was decreased and C50-lycopene was increased in the case of using CrtI_(N304P) as compared to the case of using CrtI-m2 (FIG. 10).

(Example 8) Study on Use of CrtW and CrtZ Derived from Brevundimonas sp. Strain SD-212

In Example 5, it was attempted to produce C50-astaxanthin, in addition to C50-β-carotene, by the use of CrtW and CrtZ derived from Paracoccus sp. strain N81106. A polar peak appeared, but most of C50-β-carotene remained without any modification by CrtW or CrtZ. Thus, the synthesis of C50-astaxanthin and its intermediates C50-zeaxanthin and C50-canthaxanthin was attempted by the use of CrtW and CrtZ having higher efficiency.

As previously reported (Choi S. et al., Mar Biotechnol 7, 515-522 (2005), Choi S. et al., Appl Microbiol Biotechnol 72, 1238-1246 (2006)), it has been found that CrtW and CrtZ derived from Brevundimonas sp. strain SD-212 are a β-carotene ketolase and β-carotene hydroxylase having high efficiency, respectively. In the synthesis of a natural (C40 type) astaxanthin, the enzymes are the best choices that are currently available. Thus, the total synthesis of those genes was performed with codon optimization for E. coli, which was commissioned to DNA2.0. Through the use of those genes, pUC-pBAD-CrtI_(N304P)-CrtY-CrtW_(BD)-CrtZ_(BD) was prepared. Further, pUC-pBAD-CrtI-m2-CrtY-CrtW_(BD) and pUC-pBAD-CrtI-m2-CrtY-CrtZ_(BD) were also prepared. The plasmid maps of pUC-pBAD-CrtI-m2-CrtY-CrtW_(BD), pUC-pBAD-CrtI-m2-CrtY-CrtZ_(BD), and pUC-pBAD-CrtI_(N304P)-CrtY-CrtW_(BD)-CrtZ_(BD) are illustrated in FIGS. 11A, 11B, and 11C, respectively, and the nucleotide sequences thereof are set forth in SEQ ID NOS: 16, 17, and 18, respectively.

Those plasmids were introduced into E. coli (XL1-Blue) together with pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A), and culturing and extraction were performed by the method shown in Example 3. The obtained carotenoid extracts were each injected into an HPLC-photodiodearray system. Analysis was performed at a flow rate of 1 mL/min by using a μBondapak column (100×8 mm, RCM-type, Waters) as a column and using methanol as an eluent.

As a result, C50-zeaxanthin was specifically synthesized in pUC-pBAD-CrtI-m2-CrtY-CrtZ_(BD) (panel a in FIG. 12), and C50-canthaxanthin was specifically synthesized in the case of using pUC-pBAD-CrtI-m2-CrtY-CrtW_(BD) (panel b in FIG. 12). Further, C50-astaxanthin was specifically synthesized in the case of using pUC-pBAD-CrtI_(N304P)-CrtY-CrtW_(BD)-CrtZ_(BD) (panel c in FIG. 12).

(Example 9) Increase in C50-Astaxanthin Amount by Co-Expression of Idi

C50-astaxanthin was synthesized in E. coli by the method described in Example 8 using pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A) and pUC-pBAD-CrtI_(N304P)-CrtY-CrtW_(BD)-CrtZ_(BD), and a synthesis amount thereof was determined from an HPLC peak area thereof.

As a result, C50-astaxanthin was synthesized at 288 μg/gDCW (FIG. 13).

In order to co-express idi therewith, pAC-crtM_(F26A,W38A,F233S)-fds_(Y81A,V157A)-idi was prepared and transformed into E. coli (XL1-Blue) together with pUC-pBAD-CrtI_(N304P)-CrtY-CrtW_(BD)-CrtZ_(BD). Then, a carotenoid synthesis amount was determined by a similar method. The plasmid map of pAC-CrtM_(F26A,W38A,F233S)-fds_(Y81A,V157A)-idi is illustrated in FIG. 11D and the nucleotide sequence thereof is set forth in SEQ ID NO: 19.

As a result, C50-astaxanthin was synthesized at 884 μg/gDCW (FIG. 13).

Example 10

A compound in the peak of C50-β-carotene obtained in Example 4 (peak 7 in FIG. 6D) and compounds in the peaks of C50-zeaxanthin, C50-canthaxanthin, and C50-astaxanthin identified in Example 8 (peaks 1, 2, and 3 in FIG. 12) were identified.

The maximum absorption wavelengths of the compound in the peak (peak 7 in FIG. 6D) of C50-β-carotene were 467 (shoulder), 501, and 534 nm in methanol, which was shifted to the lower wavelength side than those of C50-lycopene. Thus, it is apparent that the compound is a carotenoid having a ring structure (Takaichi, S. & Shimada, K. Methods Enzymol. 213, 374-385 (1992)). Further, the prolonged elution time in reverse phase HPLC also indicates that cyclization occurred. The molecular mass was 668, which was identical to a value calculated for C50-β-carotene.

The compound in the peak (peak 3 in FIG. 12) of 50-astaxanthin gave a broad absorption spectrum having a maximum absorption wavelength at 512 nm in methanol. The molecular mass was 728, which was consistent with a value predicted from the structural formula of C50-astaxanthin. In addition, it was attempted to determine a partial structure thereof by chemical derivatization in accordance with the previous report (S. Takaichi et al., Org. Mass Spdectroscopy, 28, 785-788 (1993)). This compound, when subjected to reduction with NaBH₄, gave the same absorption spectrum as that of C50-β-carotene, and had a molecular mass increased to 732. This revealed the presence of two carbonyl groups at the 4,4′-positions. When the compound before NABH₄ treatment was used to prepare its diacetyl and ditrimethylsilyl derivatives, the molecular masses were 812 and 872, respectively. The increases correspond to increases in molecular weight of two acetyl groups and two trimethylsilyl groups, respectively. This revealed the presence of two hydroxyl groups. On the other hand, when the compound after NABH₄ treatment was used to prepare its diacetyl and ditrimethylsilyl derivatives in the same manner as described above, the molecular masses were 900 and 1,020, respectively. In other words, the presence of four hydroxyl groups could be confirmed. In addition, when the compound was purified and subjected to NMR analysis, the compound was clearly identified as C50-astaxanthin (FIG. 14).

The compound in the peak (peak 1 in FIG. 12) of C50-zeaxanthin had a molecular mass of 700, which HPLC elution profile and absorption spectrum were as shown in panel (a) of FIG. 12. The compound was determined as C50-zeaxanthin by considering the shift of its elution peak to the polar side and so on.

The compound in the peak (peak 2 in FIG. 12) of C50-canthaxanthin had a molecular mass of 696, which HPLC elution profile and absorption spectrum were as shown in panel (b) of FIG. 12. Thus, the compound was identified as C50-canthaxanthin in consideration of, for example, the shift of its elution peak to the polar side.

(Example 11) Production of Carotenoids Having Great Variety of Structures by Additional Expression of CrtG and CrtX

A study on the diversification of carotenoid structures to be synthesized was made by the additional expression of a crtG gene derived from Brevundimonas sp. strain SD-212 (Nishida, Y. et al, Appl Environ Microbiol 71, 4286-4296, 2005) and a crtX gene derived from Pantoea ananatis. The crtX gene is a gene encoding a zeaxanthin glucosyl transferase.

First, pUC-pBAD-CrtI_(N304P)-CrtY-CrtG-CrtZ_(BD) and pUC-pBAD-CrtI_(N304P)-CrtY-CrtG-CrtW_(BD)-CrtZ_(BD) were prepared by using the crtG gene derived from Brevundimonas sp. strain SD-212. The plasmid maps of pUC-pBAD-CrtI_(N304P)-CrtY-CrtG-CrtZ_(BD) and pUC-pBAD-CrtI_(N304P)-CrtY-CrtG-CrtW_(BD)-CrtZ_(BD) are illustrated in FIGS. 15A and 15B, respectively, and the nucleotide sequences thereof are set forth in SEQ ID NOS: 20 and 21, respectively.

In the case of culturing E. coli co-expressing pUC-pBAD-CrtI_(N304P)-CrtY-CrtG-CrtZ_(BD) and pAC-CrtM_(F26A,W38A,F233S)-FDS_(Y81A,V157A), C₅₀-caloxanthin and C₅₀-nostoxanthin, which are hydroxylated zeaxanthin at the 2-position and 2′-position, can be synthesized (FIG. 16).

In the case of culturing E. coli co-expressing pUC-pBAD-CrtI_(N304P)-CrtY-CrtG-CrtW_(BD)-CrtZ_(BD) and pAC-CrtM_(F26A,W38A,F233S)-FDS_(Y81A,V157A), C₅₀-2-hydroxyastaxanthin and C₅₀-2,3,2′,3′-tetrahydroxy-β,β-carotene-4,4′-dione, which are hydroxylated astaxanthin at the 2-position and 2′-position, can be synthesized (FIG. 16).

Further, it is also possible to synthesize C₅₀-2-hydroxycanthaxanthin and C₅₀-2,2′-dihidroxycanthaxanthin, which are hydroxylated canthaxanthin at the 2-position and 2′-position (FIG. 16).

Next, pUC-pBAD-CrtI_(N304P)-CrtY-CrtX-CrtZ_(BD) and pUC-pBAD-CrtI_(N304P)-CrtY-CrtX-CrtW_(BD)-CrtZ_(BD) were prepared by using the crtX gene derived from Pantoea ananatis. The plasmid maps of pUC-pBAD-CrtI_(N304P)-CrtY-CrtX-CrtZ_(BD) and pUC-pBAD-CrtI_(N304P)-CrtY-CrtX-CrtW_(BD)-CrtZ_(BD) are illustrated in FIGS. 15C and 15D, respectively, and the nucleotide sequences thereof are set forth in SEQ ID NOS: 22 and 23, respectively.

In the case of culturing E. coli co-expressing pUC-pBAD-CrtI_(N304P)-CrtY-CrtX-CrtZ_(BD) and pAC-CrtM_(F26A,W38A,F233S)-FDS_(Y81A,V157A), C₅₀-zeaxanthin-β-D-glucoside and C₅₀-zeaxanthin-β-D-diglucoside, which are glycosidated zeaxanthin at the hydroxyl groups at the 3-position and 3′-position, can be synthesized (FIG. 17).

In the case of culturing E. coli co-expressing pUC-pBAD-CrtI_(N304P)-CrtY-CrtX-CrtW_(BD)-CrtZ_(BD) and pAC-CrtM_(F26A,W38A,F233S)-FDS_(Y81A,V157A), C₅₀-astaxanthin-β-D-glucoside and C₅₀-astaxanthin-β-D-diglucoside, which are glycosidated astaxanthin at the hydroxyl groups at the 3-position and 3′-position, can be synthesized (FIG. 17).

In fact, when E. coli (XL1-Blue) co-expressing any one of pUC-pBAD-CrtI_(N304P)-CrtY-CrtG-CrtZ_(BD), pUC-pBAD-CrtI_(N304P)-CrtY-CrtX-CrtZ_(BD), pUC-pBAD-CrtI_(N304P)-CrtY-CrtG-CrtW-CrtZ_(BD), and pUC-pBAD-CrtI_(N304P)-CrtY-CrtX-CrtW_(BD)-CrtZ_(BD) together with pAC-CrtM_(F26A,W38A,F233S)-FDS_(Y81A,V157A) was spread on an LB solid medium to form colonies, the formed colonies developed a reddish violet color (FIG. 18).

The colonies were inoculated into 2 mL of an LB liquid medium and cultured for 16 hours. After that, the culture medium was inoculated into 30 mL of a TB liquid medium in a 1/100 volume with respect to the TB liquid medium. After incubation under rotation at 200 rpm at 30° C. for 36 hours, L-arabinose was added so as to have a final concentration of 0.2% (v/v), followed by incubation for an additional 36 hours. The cells were harvested and collected from 2 mL of the culture medium and washed with physiological saline, and then a carotenoid fraction was extracted with the addition of 1 mL of acetone. FIG. 18 shows the cell pellets and the acetone extracts. Further, FIGS. 19A and 19B show the absorption spectra of the respective extracts.

Next, E. coli (XL1-Blue) was transformed with pUC-pBAD-CrtI_(N304P)-CrtY-CrtX-CrtZ_(BD) or pUC-pBAD-CrtI_(N304P)-CrtY-CrtG-CrtZ_(BD) and pAC-CrtM_(F26A,W38A,F233S)-FDS_(Y81A,V157A). The cell colonies were inoculated into 2 mL of an LB liquid medium and cultured for 16 hours. After that, the culture medium was inoculated into 30 mL of a TB liquid medium in a 1/100 volume with respect to the TB liquid medium. After incubation under rotation at 200 rpm at 30° C. for 36 hours, L-arabinose was added so as to have a final concentration of 0.2% (v/v), followed by incubation for an additional 36 hours. The cells were harvested by centrifuging the culture medium and washed with physiological saline. 10 mL of acetone were added to the cell pellet to extract a carotenoid fraction. 1 mL of chloroform and 35 mL of 10% NaCl were added to the acetone extract to extract the carotenoid fraction into a chloroform phase. All the chloroform extracts were collected and dehydrated with the addition of MgSO₄. The chloroform solvent was eliminated by nitrogen, and the carotenoid fraction was finally concentrated into 100 μL of methanol/THF (1:1, v/v). Thus, a carotenoid extract was obtained. 10 μL (10% of the total volume, corresponding to 3 mL of the medium) of the obtained carotenoid extract were injected into an HPLC-photodiodearray system. HPLC analysis was performed in accordance with the conditions of Nishida, Y. et al, Appl Environ Microbiol, 71, 4286-4296 (2005) (column: TSK gel ODS-80 Ts column (4.6-mm inner diameter by 150 mm; Tosoh Co.), eluent: Eluent A (methanol/water, 95:5) for 5 minutes, Eluent A to Eluent B (methanol/tetrahydrofuran, 7:3) for 5 minutes, Eluent B for 15 minutes, 1 mL/min, detector: photodiode array (190 to 800 nm)).

As a result, in the case of co-expression of CrtX with C50-zeaxanthin, one new peak appeared at a shorter elution time (peak in FIG. 20). A compound in the peak is estimated to be C50-zeaxanthin-β-D-diglucoside because of having the same absorption spectrum as that of C50-zeaxanthin and having higher polarity than that of C50-zeaxanthin.

Further, in the case of co-expression of CrtG with C50-zeaxanthin, two new peaks appeared (peaks 2 and 3 in FIG. 20). Compounds in the peaks are estimated to be C50-caloxanthin (peak 3 in FIG. 20) and C50-nostoxanthin (peak 2 in FIG. 20) because of, for example, having the same absorption spectrum as that of C50-zeaxanthin and having higher polarity.

(Example 12) Biosynthesis of C₅₅ and C₆₀ Carotenoids

A mutant FDS having introduced therein mutations I78G and Y81A (FDS_(I78G,Y81A)) synthesizes C₂₅PP and a prenyl diphosphate (e.g., C₃₀PP or C₃₅PP) which is a resultant of further addition of C₅ unit(s) to C₂₅PP (Ohnuma S et al., J Biol Chem 273, 26705-26713, 1998). Further, a C₃₀PP synthase (HexPS) derived from Micrococcus luteus synthesizes C₃₀PP (Shimizu N et al, J Bacteriol 180, 1578-1581, 1998). The biosynthesis of a larger carotenoid backbone than a C₅₀ backbone was attempted by co-expressing FDS_(I78G,Y81A) or HexPS with a CrtM variant.

First, pAC-hexPS, pAC-FDS_(I78G,Y81A)-idi, and pUC-CrtM_(F26A,W38A,F233S) were prepared. The plasmid maps of pAC-hexPS, pAC-FDS_(I78G,Y81A)-idi, and pUC-CrtM_(F26A,W38A,F233S) are illustrated in FIGS. 21A, 21B, and 21C, respectively, and the nucleotide sequences thereof are set forth in SEQ ID NOS: 24, 25, and 26, respectively.

E. coli (XL1-Blue) was transformed with pAC-FDS_(I78G,Y81A)-idi or pAC-hexPS together with pUC-CrtM_(F26A,W38A) or pUC-CrtM_(F26A,W38A,F233S). The culturing of the cells and the analysis of the produced carotenoid were performed in the same manner as in Reference Example 1.

FIG. 22 shows the results. In the case of using CrtMF26A,W38A,F233S, a C₅₅ carotenoid was produced in addition to C₄₅ and C₅₀ carotenoids under co-expression of pAC-FDS_(I78G,Y81A)-idi therewith. Further, a C₆₀ carotenoid was specifically synthesized under co-expression of hexPS therewith. On the other hand, in the case of using CrtM_(F26A,W38A), it was impossible to confirm the production of any of the C₅₅ carotenoid and the C₆₀ carotenoid. Accordingly, the F233S mutation of CrtM is essential for the production of the C₅₅ carotenoid and the C₆₀ carotenoid.

Next, pAC-FDS_(I78G,Y81A)-idi or pAC-hexPS was co-expressed with 8 kinds of CrtM variants (pUC-CrtM variants), and carotenoid synthesis amounts thereof were investigated by a similar method. FIG. 23 shows the results. The C₅₅ backbone was synthesized in a larger amount in the cells co-expressing FDS_(I78G,Y81A)-idi with CrtM_(F26A,F233S) or CrtM_(F26A,W38A,F233S). The C₆₀ backbone was efficiently synthesized in the cells co-expressing HexPS with CrtM_(W38A,F233S) or CrtM_(F26A,W38A,F233S).

INDUSTRIAL APPLICABILITY

As described above, the production method of the present invention can synthesize the desaturated C₅₀ carotenoid in an extremely efficient manner, and allows various C_(H) carotenoids to be synthesized. The C₅₀ carotenoid is rarely found in nature, and it is not too much to say that there is no synthesis example thereof. Carotenoids are known to have physiological activities such as an antioxidant action. Of those, the C₅₀ carotenoid is expected to exhibit unprecedented novel actions and to have remarkably enhanced activities as compared to conventional carotenoids. For example, the C₅₀ carotenoid is expected to have the potentiality of having a high antioxidant activity, an application as a novel seed for a physiologically active substance having an antitumor activity or the like, and a use as a functional pigment molecule. Further, the production method of the present invention may be utilized in the highly efficient synthesis of the desaturated C₅₅ carotenoid, the desaturated C₆₀ carotenoid, and the like. In addition, the production method of the present invention is considered to drastically increase variety of synthesizable carotenoids, by being used in combination with related art such as cells having an enhanced isoprenoid synthetic pathway (Klein-Marcuschamer D et al.: Trends Biotechnol 25, 417-424 (2007), Kirby J et al.: Nat Prod Rep 25, 656-661 (2008)). 

1-15. (canceled)
 16. A method of producing a carotenoid having 55 carbon atoms, comprising: culturing, in a medium, a cell transformed with a mutant diapophytoene synthase gene and a mutant farnesyl diphosphate synthase gene; and obtaining the carotenoid having 55 carbon atoms from a culture after the culturing, wherein the transformation is performed by a plasmid having a nucleotide sequence of SEQ ID NO: 26 that contains the mutant diapophytoene synthase gene and a plasmid having a nucleotide sequence of SEQ ID NO: 25 that contains the mutant farnesyl diphosphate synthase gene.
 17. A method of producing a carotenoid having 60 carbon atoms, comprising: culturing, in a medium, a cell transformed with a mutant diapophytoene synthase gene and a mutant hexaprenyl diphosphate synthase gene; and obtaining the carotenoid having 60 carbon atoms from a culture after the culturing, wherein the transformation is performed by a plasmid having a nucleotide sequence of SEQ ID NO: 26 that contains the mutant diapophytoene synthase gene and a plasmid having a nucleotide sequence of SEQ ID NO: 24 that contains the mutant farnesyl diphosphate synthase gene. 